Conjugation of biomolecules using diels-alder cycloaddition

ABSTRACT

A method is provided for covalently linking carbohydrates, proteins, nucleic acids, and other biomolecules under neutral conditions, using a Diels-Alder cycloaddition reaction. In an example, activated carbon-carbon double bonds were attached to free amino sites of a carrier protein, and a conjugated diene was attached to a carbohydrate hapten. Spontaneous coupling of the carbohydrate and the protein components under very mild conditions provided glycoconjugates containing up to 37 carbohydrate hapten units per carrier protein molecule. The method is also applicable to the immobilization of biomolecules on gel or solid supports. The conjugated products are useful as immunogens and as analytical and diagnostic reagents.

PRIORITY CLAIM

This application is a divisional of U.S. patent application Ser. No.09/919,637, filed Aug. 1, 2001, now U.S. Pat. No. 6,673,905 which claimsthe benefit of U.S. Provisional Patent Application No. 60/223,959, filedAug. 9, 2000. Both of the prior applications are incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The invention is in the filed of bioorganic chemistry, more specificallythe field of conjugation of biomolecules. The conjugated productsprepared by the methods of the invention are useful, for example, asinoculants for the generation of antibodies, and as vaccines. Themethods of the invention may also be used to immobilize biomolecules onsolid supports. The immobilized biomolecules are useful in many fields,such as for example catalysis, separation, analysis, and diagnostics.

BACKGROUND

The conjugation of biomolecules to solid and gel supports is a commonoperation in may laboratories, and many methods have been developed forthis purpose. Immobilization of enzymes (I. Chernukhin, E. Klenova,Anal. Biochem. 2000, 280:178–81), oligonucleotides (J. Andreadis; L.Chrisey, Nucleic Acids Res. 2000, 28:e5; A. Drobyshev et al., Nucl.Acids. Res. 1999, 27:4100–4105), antibodies (P. Soltys, M. Etzel,Biomaterials 2000, 21:37–48), and antigens (M. Oshima, M. Atassi,Immunol. Invest. 1989, 18:841–851) on solid and gel supports enables thepreparation of useful products such as chromatographic media (Meth.Enzym. W. Jakoby, M. Wilchek, eds., 1974, 34, Academic Press, NY),catalysts (T. Krogh et al., Anal. Biochem., 1999, 274:153–62),biosensors (J. Spiker, K. Kang, Biotechnol. Bioeng. 1999, 66:158–63),and numerous diagnostic (G. Ramsay, Nature Biotechnol., 1998, 16:40–44)and research tools (C. Bieri et al., Nature Biotechnol. 1999,17:1105–1108). Even whole cells may be immobilized by such methods (E.Olivares, W. Malaisse, Int. J. Mol. Med. 2000, 5:289–290).

The most robust form of attachment of a biomolecule to a surface orother support is via covalent bonds. Typically, such bonds areheteroatom-based (e.g., amide, ester, and disulfide bonds), because suchbonds are easily formed under mild conditions. Non-covalent attachmentvia specific binding pairs (e.g., biotin-avidin or antibody-antigeninteractions) is also commonly employed, but such methods still requireinitial conjugation of the specific binding pairs to the biomolecule andsupport. The use of carbon-carbon bonds for this purpose is very rare,because formation of C—C bonds is more difficult, especially under themild aqueous conditions appropriate for working with proteins.

The use of the Diels-Alder reaction to attach a member of a specificbinding pair has been described. In this report (M. N. Yousaf and M.Mrksich, J. Am. Chem. Soc., 1999, 121:4286), a Diels-Alder reaction wasused to covalently attach a biotinylated diene to an immobilizeddienophile, and the immobilized biotin was subsequently used tonon-covalently immobilize streptavidin. Thee workers have more recentlyused a Diels-Alder reaction to immobilize the peptide RGDS on aself-assembled alkanethiol monolayer on a gold surface (M. N. Yousaf, B.T. Houseman, M. Mrksich, Angew. Chem. Int. Ed. Engl., 2001, 40:1093).The use of the Dials-Alder reaction to effect the actual covalentcoupling or immobilization event of large biomolecules, however, had notpreviously been described.

The conjugation of biomolecules to one another is likewise a very commonprocedure, and is subject to most of the concerns and limitationsdescribed above for biomolecule immobilization. Covalent attachment ofhaptens to proteins has been a target of synthetic endavors since thediscovery by Landsteiner that this process can convert non-immunogenicmolecules to immunogenic material (K. Landsteiner, H. Lampl, Biochem.Zeitschr. 1918, 86:343). The application of this concept tocarbohydrates by Goebel and Avery revealed that covalentcarbohydrate-protein conjugates are immunogenic and can generateanti-carbohydrate antibodies (W. Goebel, J. Exp. Med. 1940, 72:33). Theuse of Landsteiner's principle has led to the development ofcarbohydrate-protein conjugates that are valuable tools in glycomedicalresearch, and that are useful as pharmaceuticals. In particular, proteinconjugates of fragments of the capsular polysaccharide of Haemophilusinfluenzae type b have become established as successful vaccines (J.Robbins et al., J. Am. Med. Assoc. 1996, 276:1181). Several otherbacterial saccharide-protein conjugates are in various stages ofclinical studies (E. Kondau et al., J. Infect. Dis. 1998, 177:383–387;E. Konadu et al., Infect. Immun. 2000, 68:1529–1534) while numerousothers are in the pre-clinical phase (V. Pozagay et al., Proc. Natl.Acad. Sci. USA 1999, 96:5194).

The choice of methods for covalent bond formation between biomoleculessuch as carbohydrates and proteins is restricted by their limitedsolubility in organic solvents, and in many cases by their pH andtemperature sensitivity. In almost all cases, water is the only solventthat can be used for conjugation of carbohydrates or proteins, and theconditions are usually limited to temperatures under 50° C. and pHvalues between 6 and 8.

Numerous methods have been developed for the attachment ofpolysaccharides to proteins (C. Peeters et al., in Vaccine Protocols, A.Robinson et al., Eds., 1996 Humana Press, NJ, p. 111; W. Dick, Jr., M.Beurret, in Contrib. Microbiol. Immunol., J. Cruse and R. Lewis, eds.,1989, 10:48–114, Karger, Basel; H. Jennings, R. Sood, inNeoglycoconjugates. Preparation and Applications. Y. Lee, R. Lee, eds.,Academic Press, New York, 1994, p. 325). However, only a few of thesemethods are capable of coupling oligosaccharides to carriers in asite-selective fashion. Most prominent among these is reductiveamination, which converts the reducing-end residue of the polysaccharideinto a polyhydroxy alkylamino moiety, which unfortunately causes theloss of this unit as a true carbohydrate in the resulting glycoconjugate(V. Pozgay, Glycoconjugate J. 1993, 10:133).

This problem can be solved by chemical synthesis of oligosaccharideglycosides with aglycons that bear a (latent) reactive group. Examplesinclude alkenyl groups (M. Nashed, Carbohydr. Res. 1983, 123:241–246; J.Allen, S. Danishefsky, J. Am. Chem. Soc. 1999, 121:10875), 3-aminopropyl(G. Veeneman et al., Tetrahedron 1989, 45:7433), 4-aminophenylethyl (R.Eby, Carbohydr. Res. 1979, 70:75), 4-aminophenyl (S. Stirm et al.,Justus Liebigs Ann. Chem. 1966, 696:180), 6-aminohexyl (J. Hermans etal., Rec. Trav. Chim. Pays-Bas 1987, 106:498; R. Lee et al.,Biochemistry 1989, 28:1856), 5-methoxycarbonylpentyl (S. Sabesan, J.Paulson, J. Am. Chem. Soc. 1986, 108:2068; V. Pozsgay, Org. Chem. 1998,63:5983), 8-methoxycarbonyloctyl (R. Lemieux et al., J. Am. Chem. Soc.1975, 97, 4076; B. Pinto et al., Carboydr. Res. 1991, 210, 199)4-aminobenzyl (W. Goebel, J. Exp. Med. 1940, 72:33), ω-aldehydoalkyl (V.Pozsgay, Glycoconjugate J. 1993, 10:133), 3-(2-aminoethylthio)propyl (Y.Lee, R. Lee, Carbohydr. Res. 1974, 37:193), 2-chloroethylthiogly (M.Ticha et al., Glycoconjugate J. 1996, 13:681) and 1-O-succinimidederivatives (M. Andersson, S. Oscarson, Bioconjugate Chem. 1993, 4:246;B. Davis, J. Chem. Soc. Perkin I 1999, 3215).

These aglycons introduce spacers that can be linked to a protein eitherdirectly or after insertion of a secondary linker. For this purpose theuse of an activated dicarboxylic acid has been reported (R. van den Berget al., Eur. J. Org. Chem. 1999, 2593–2600). In another procedure, asulfhydryl group at the terminal position of the spacer allows theformation of a disulfide bridge with proteins using the dithiopyridylmethod (J. Evenberg et al., J. Infect. Dis. 1992, 165(sup. 1):S152). Ina related protocol, a thiolated protein is coupled with amaleimido-derivatized saccharide (J. Mahoney, R. Schnaar, MethodsEnzymol. 1994, 242:17). N-acryloylamidophenyl glycosides may be coupledto unmodified proteins using a Michael addition (A. Romanowska et al.,Methods Enzymol. 1994, 242:90). As an alternative to glycosideformation, direct coupling of a carbohydrate to a linker via amide bondshas also been used (A. Fattom et al., Infect. Immun. 1992, 60:584–589),but this approach is limited to carboxyl-containing carbohydrates.

The yields of any of these methods rarely exceed 40% and are generallyin the 10–20% range (R. van den Berg et al., Eur. J. Org. Chem. 1999,2593–2600), especially when medium or high carbohydrate loading in theconjugate is attempted. This problem is compounded by the fact that theoligosaccharide haptens, usually obtained in multistep syntheses or bycontrolled degradation of polysaccharides, can rarely be reconverted intheir active or activable form after the coupling procedure. Anadditional problem with most chemical coupling methods employed to dateis the formation of cross-linked byproducts, due to the presence ofmultiple reactive functional groups (e.g., amines, acids, hydroxyls, andsulfhydryls) on most biomolecules. Avoidance of this problem requiresthat the reactive groups be blocked, with requires additional processingsteps and may alter the physicochemical and immunological properties ofthe biomolecule. Thus, there remains a need for a mild andsite-selective method for coupling biomolecules to one another, whichavoids the problems of low yields, crosslinking, and loss of startingmaterials. For similar reasons there remains a need for mild andselective methods for attaching biomolecules to surfaces and solid andgel supports.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides and experimentally simple protocol forthe convalent attachment of biomolecules to one another and to supports,that can avoid many of the above-mentioned problems. The invention makesuse of the well-known Diels-Alder cycloaddition reaction that takesplace between a double bond and conjugated diene. This reaction hastraditionally been carried out in organic solvents, but can proceed inaqueous solutions as well (R. Breslow, D. Rideout, J. Am. Chem. Soc.1980, 102:7816; A. Lubineau, J. Auge, Top. Curr. Chem. 1999, 206:1; P.Garner, in Organic Synthesis in Water, P. Gricco, ed., Blackie Academicand Professional, London, 1998, p. 1.)

Carbohydrates have been employed as chiral auxiliaries and/or watersolubilizing agents for Diels-Alder reactions, wherein a conjugateddiene system is converted to a glycoside prior to the cycloaddition (A.Lubineau et al., J. Chem. Soc. Perkin l 1997, 2863–2867; see also S.Pellegrinet, R. Spanevello, Org. Lett. 2000, 2:1073–1076). As notedabove, the Diels-Alder reaction has also been used to covalently attachbiotin to a support (M. N. Yousaf and M. Mrksich, J. Am. Chem. Soc.,1999, 121:4286). However, the Diels-Alder reaction has not previouslybeen extended to the direct covalent conjugation of biopolymers or othertypes of polymeric materials. Among the advantages of the method of theinvention are the mild and neutral conditions, good yields, negligiblecross-linking, and facile recovery of excess and/or unreactedbiomolecules in their conjugatable form.

The invention also provides conjugated biomolecules, which are useful asimmunostimulatory agents for production of antibodies and induction ofimmunity, methods of inducing antibody production with the conjugatedbiomolecules, and vaccine compositions comprising the conjugatedbiomolecules.

The invention also provides polyclonal and monoclonal antibodiesgenerated by administration of the conjugated biomolecules to a mammal,and methods of using the induced antibodies for inducing passiveimmunity. The antibodies are useful of therapeutic, diagnostic, andanalytical purposes.

The invention also provides immobilized biomolecules, and methods fortheir preparation, which are useful in many areas, such aschromatographic media, catalysts, components of diagnostic devices,biosensors, and as research tools.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the increase in molecular weight as a function oftime of a conjugate (conjugate 28) of the invention relative to the massof the core protein with the dienophiles attached.

FIG. 2 illustrates a similar time-dependent experiment as represented inFIG. 1, conducted with the same maleimido-derivatized protein, usingpreviously-described diene 8.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a new method for conjugation of biomoleculesbased on the Diels-Alder cycloaddition reaction. The technique involvesthe introduction of an activated double bond into a first biomoleculecomponent, and a conjugated diene into a second biomolecule component,which are to be covalently linked together. The diene- anddienophile-modified biomolecules may then be purified to the extenddesired. The two components are then simply combined under neutralconditions, and the cycloaddition reaction is allowed to proceed.

As used herein, the term “biomolecule” refers generally to the large,complex molecules produced within living cells, and to synthetic andsimi-synthetic analogues thereof. Examples include proteins, peptides,oligo- and poly-saccharides, and oligo- and poly-nucleic acids, andvarious combinations thereof such as for example glycoproteins andnucleoproteins. Larger complexes, such as ribosomes, cellularsubstructures, and even entire cells are intended to fall within themeaning of the term as well.

In certain of the examples presented, the diene moiety is introducedinto a carbohydrate component and the activated double bond into apolypeptide component. This may be reversed if desired, as a matter ofconvenience or if required by the synthetic design, as shown in anotherof the examples. The cycloaddition step proceeds in most cases underneutral conditions, at or below physiological temperatures. Where theyare of sufficiently low molecular weight, unreacted components can berecovered for re-use by simple diafiltration of the coupling reactionmixture.

The embodiment of the invention disclosed in certain of the examplesbelow incorporates an electron-deficient carbon-carbon double bound intoa protein, with human serum albumin (HSA) being used as an example andthe commercially available reagent 3-sulfosuccinimidyl4-maleimidobutyrate being used as the reagent. In these embodiments, thediene component of the Diels-Alder reaction is incorporated into acarbohydrate, with derivatives of trans,trans-hexa-2,4-dien-1-ol,1-amino-hexa-2,4-diene, and octa-2,4-dienoic acid hydrazide being usedas dienes. It will be understood that in general, the Diels-Alderreaction will occur between sterically accessible dienes and dienophilesregardless of the nature of the attached biomolecules, and that byappropriate selection of reagents both homoconjugates andheteroconjugates of proteins, carbohydrates, and oligonucleotides can becarried out by the methods of this invention.

The methods of the invention unexpectedly provide for a degree ofcontrol over the rate of coupling of the diene and dienophilecomponents, simply by modification of the linker moieties. The molecularweight of the conjugate approaches a limiting value as the couplingreaction proceeds, which depends on the mass of the carbohydrate beingattached. The observed increase in molecular weight as a function oftime may therefore be fit to a pseudo-first order reaction kineticequation of the formΔmw=Δmw _(max)(1−e ^(−kt))where Δmw is the increase in molecular weight, Δmw_(max) is the maximumincrease that could be obtained if all the protein-linked startingmaterial were to react, k is the rate constant, and t is time. Thecurves drawn through the data of FIGS. 1 and 2 (see Example 5 for theexperimental details) were obtained by fitting the parameters of thisequation to the data. The results of this fitting are summarized inTable 2.

The faster incorporation of construct 27 relative to 8 is an interestingobservation. The observed difference apparently correlates with thedistance between the hydrophilic rhamnose moiety and the hydrophobicdiene part of the molecule. According to Breslow, the acceleratingeffect of wear on the rate of Diels-Alder reactions is due tohydrophobic pacing of the reactants (R. Breslow, D. Rideout, J. Am.Chem. Soc. 1980, 102:7816). Such an effect should be more pronounced incompound 27, where the diene sector is more isolated from thehydrophilic portion, than in compound 8.

The Diels-Alder reaction requires a highly organized transition state,and the ability of the components of the present invention to achievethe proper geometry for cycloaddition despite the mass and bulk of theattached biomolecules is remarkable.

In a preferred embodiment of the invention, one of the biomolecules tobe linked is a hapten or antigen, and the other is a carrier. In aparticularly preferred embodiment, the hapten or antigen is apolysaccharide moiety. Examples of antigenic polysaccharides are thecapsular polysaccharides of Haemophilus influenzae type b, Neisseriameningitidis, Group B Streptococci, Salmonella typhi, E. coli, andPneumococci.

Carriers are chosen to increase the immunogenicity of the hapten orantigen, and/or to raise antibodies against the carrier itself which maybe medically beneficial. Carriers that fulfill these criteria are knownin the art (see, e.g., A. Fattom et al., Infect. Immun. 1990, 58,2309–2312; Devi, J. Robbins, R. Schneerson, Proc. Natl. Acad. Sci. USA1991, 88:7175–7179; S. Szu, X el al., Infect. Immun., 1991,59:4555–4561; S. Szu et al., J. Exp. Med., 1987, 166:1510–1524). Acarrier can be a natural, semi-synthetic, or synthetic materialcontaining one or more functional groups, for example primary and/orsecondary amino groups, azido groups, hydroxyl groups, or carboxylgroups, to which a diene or dieneophile Diels-Alder reactant moiety canbe attached. The carrier can be water soluble or insoluble, and ispreferably a polypeptide.

Examples of water soluble polypeptide carriers include, but are notlimited to, natural, synthetic, or semisynthetic peptides or proteinsfrom bacteria or viruses, e.g., bacterial, bacterial outer membraneproteins, bacterial toxins and toxoids such as tetanus toxin/toxoid,diphtheia toxin/toxoid, Pseudomonas aeruginosa exotoxin/toxoid/protein,pertussis toxin/toxoid, and Clostridium perfringens exotoxins/toxoid.Viral proteins such as hepatitis B surface antigen and core antigen mayalso be used as carriers, as well as proteins from higher organisms suchas keyhole limpet hemoxyanin, horseshoe crab hemocyanin, edestin,mammalian serum albumins, mammalian ganmma-globulins, and IgG.

Polysaccharide carriers include, but are not limited to, dextran,capsular polysaccharides from microorganisms such as the Vi capsularpolysaccharide from S. typhi, which is described in U.S. Pat. No.5,204,098, (incorporated by reference herein); Pneumococcus group 12(12F and 12A) polysaccharides; Haemophilus influenzae type dpolysaccharide; and certain plant, fruit, or synthetic oligo- orpolysaccharides which are immunologically similar to capsularpolysaccharides, such as pectin, D-glacturonan, oligogalacturonate, orpolygalacturonate, for example as described in U.S. Pat. No. 5,738,855(incorporated by reference herein).

Examples of water insoluble carriers include, but are not limited to,aminoalkyl agarose, e.g., aminopropyl or aminohexyl SEPHAROSE (PharmaciaInc., Piscataway, N.J.), aminopropyl glass, cross-linked dextran, andthe like, to which a diene or dienophile can be attached. Other carriersmay be used provided that a functional group is available for covalentlyattaching a diene or dienophile.

Examples of dienophiles include, but are not limited to, maleimides,acrylamides, azodicarboxylates, quinones, and1,2,4-triazoline-3,5-diones. Examples of dienes include, but are notlimited to, esters and glycosides of hexa-2,4-dien-1-ol,penta-2,4-dien-1-ol, furan-2-methanol, and furan-1-methanol; esters,amides, and hydrazides of octa-2,4-dienoic acid; and amides of1-aminohexa-2,4-diene and 1- and 2-aminomethylfuran. The above-mentionedamines may also be coupled with aldehydo-biomolecules via reductiveamination, and hydrazides may be attached to such biomolecules viacondensation.

The invention also provides biomolecule conjugates of general formulas Iand II below:

where R and R′ are independently H or methyl, or together constituteCH₂, CH₂CH₂, SO₂, or O; X is CH or N; Y is N, CH═C, or NH—N; and B₁ andB₂ comprise biomolecules independently selected from the groupconsisting of polypeptides, carbohydrates, polysaccharides, and nucleicacids, and are optionally attached via a linker.

The invention also provides immobilized biomolecules of formulas I andII above, wherein one of B₁ and B₂ may be a solid or gel support.Examples of solid supports include, but are not limited to,aminopropylsilylated glass and silica surfaces, gold surfacesfunctionalized with thiol-bound linkers, functionalized macroporouspolystyrene beads, and surface-derivatized microtiter plate wells.Examples of gel supports include, but are not limited to, functionalizedagarose gels such as cyanogen bromide activated agarose, aminoethylagarose, and carboxymethyl agarose.

The formulas above are intended to indicate that the group B₁ may beattached alpha or beta to the group R′ as shown below:

There are many known methods of attachment of small molecules tobiomolecules, there are many known linker moieties for attachment ofchemical moieties to biomolecules, and there are many known dienes anddienophiles that readily take part in cycloaddition reaction at or nearroom temperature. Those skilled in the art will thus appreciate thatthere are many obvious combinations of attachment methods, linkers, anddiene and dienophile partners that may be employed in the method ofbiomolecule coupling disclosed herein, which are equivalent to theexamples provided. Such modifications of the disclosed methods andresulting compositions are intended to be within the scope and spirit ofthe present invention.

It is another object of the invention to provide methods of using thepolysaccharide-carrier conjugates of this invention for eliciting animmunogenic response in mammals, including but not limited to responseswhich provide protection against, or reduce the severity of, bacterialand viral infections. The pharmaceutical compositions of this inventionare expected to be capable, upon injection into a mammal, of inducingserum antibodies against the polysaccharide component of the conjugate.

The invention also provides methods of using such conjugates, and/orpharmaceutical compositions comprising such conjugates, to induce inmammals, in particular, humans, the production of antibodies whichimmunoreact with the polysaccharide component of the conjugates.Antibodies which immunoreact with a bacterial or viral polysaccharideare useful for the identification or detection of microorganismsexpressing the polysaccharide, and/or for diagnosis of infection.Antibodies against the polysaccharide may be useful in increasingresistance to, preventing, ameliorating, and/or treating illnessescaused by microorganisms or viruses that express the polysaccharide.

The compositions of this invention are intended for active immunizationfor prevention of infection, and for preparation of immune antibodies.The compositions of this invention are designed to induce antibodiesspecific to microorganisms expressing the polysaccharide component ofthe conjugate, and to confer specific immunity against infection withsuch microorganisms.

This invention also provides compositions, including but not limited to,mammalian serum, plasma, and immunoglobulin fractions, which containantibodies which are immunoreactive with the polysaccharide component ofthe conjugates of this invention, and which preferably also containantibodies which are immunoreactive with the protein component. Theseantibodies and antibody compositions may be useful to prevent, treat, orameliorate infection and disease caused by the microorganism. Theinvention also provides such antibodies in isolated form. The inventionfurther provides methods of inducing in mammals antibodies withimmunoreact with a polysaccharide, the methods comprising administeringto a mammal a composition of the invention.

The invention also provides monoclonal antibodies, preferably producedby hybridomas, which immunoreact with a polysaccharide. The nucleic acidsequences encoding these antibodies are obtained from a mammal in whichthe production of anti-polysaccharide antibodies has been induced byadministering a composition of the invention.

As used herein, the terms “immunoreact” and “immunoreactivity” refer tospecific binding between an antigen or antigenic determinant-containingmolecule and a molecule having an antibody combining site, such as awhole antibody molecule or a portion thereof.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules.Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and portion of animmunoglobulin molecule, including those portions known in the art asFab, Fab′, F(ab′)hd 2 and F(v), as well as chimeric antibody molecules.

Polymeric Carriers

Carriers are chosen to increase the immunogenicity of the polysaccharideand/or to raise antibodies against the carrier which are medicallybeneficial. Carriers that fulfill these criteria are well-known in theart. A polymeric carrier can be a natural or a synthetic materialcontaining one or more functional groups, for example primary and/orsecondary amino groups, azido groups, or carboxyl groups, to which adiene or dienophile component can be attached. Carriers can be watersoluble or insoluble. The examples below employ proteins as carriers.

Regardless of the precise method used to prepare the conjugate, afterthe Diels-Alder coupling reaction has been carried out the unreactedmaterials are preferably removed by routine physicochemical methods,such as for example dialysis, gel filtration or ion exchange columnchromatography, depending on the materials to b separated. The finalconjugate consists of the polysaccharide and the carrier bound through aDiels-Alder adduct.

Dosage for Vaccination

The present inoculum contains an effective, immunogenic amount of apolysaccharide-carrier conjugate. The effective amount ofpolysaccharide-carrier conjugate per unit dose sufficient to induce animmune response depends, among other things, on the immunogenicity ofthe polysaccharide, the species of mammal inoculated, the body weight ofthe mammal, and the chosen inoculation regimen, as is well known in theart. Inocula typically contain polysaccharide-carrier conjugates withconcentrations of polysaccharide from about 1 micrograms to about 500micrograms per inoculation (dose), preferably about 3 micrograms toabout 50 micrograms per dose, and most preferably about 5 micrograms to25 micrograms per dose.

The term “unit dose” as it pertains to the inocula refers to physicallydiscrete units suitable as unitary dosages for mammals, each unitcontaining a predetermined quantity of active material (polysaccharide)calculated to produce the desired immunogenic effect in association withthe required diluent.

Inocula are typically prepared in physiologically and/orpharmaceutically tolerable (acceptable) carriers, and are preferablyprepared as solutions in physiologically and/or pharmaceuticallyacceptable diluents such as water, saline, phosphate-buffered saline, orthe like, to form an aqueous pharmaceutical composition. Adjuvants, suchas aluminum hydroxide, QS-21, TiterMax™ (CytRx Corp., Norcross Ga.),Freund's complete adjuvant, Freund's incomplete adjuvant, interleukin-2,thymosin, and the like, may also be included in the compositions.

The route of inoculation may be by intramuscular or subcutaneousinjection or the like, so long as it results in eliciting antibodiesreactive against the polysaccharide component. It is anticipated that insome cases the composition can be administered orally or intranasally,for example when mucosal immunity is to be induced. In order to increasethe antibody level, a second or booster dose may be administeredapproximately 4 to 6 weeks after the initial administration. Subsequentdoses may be administered as deemed necessary by the practitioner.

Antibodies

An antibody of the present invention is typically produced by immunizinga mammal with an immunogen or vaccine containing apolysaccharide-carrier conjugate, preferably a polysaccharide-proteinconjugate, to induce in the mammal antibody molecules havingimmunospecificity for the polysaccharide component of the conjugate.Antibody molecules having immunospecificity for the protein carrier mayalso be produced. The antibody molecules may be collected from themammal and, optionally, isolated and purified by methods known in theart.

Human or humanized monoclonal antibodies are preferred, including butnot limited to those identified by phage display technology, andincluding but not limited to those made by hybridomas and by mice withhuman immune systems or human immunoglobulin genes. The antibodymolecules of the present invention may be polyclonal or monoclonal.Monoclonal antibodies may be produced by methods well-known in the art.Portions of immunoglobulin molecules, such as Fabs, may also be producedby methods know in the art.

An antibody of the present invention may be contained in blood plasma,serum, hybridoma supernatants and the like. Alternatively, theantibodies of the present invention are isolated to the extend desiredby well known techniques such as, for example, ion chromatography oraffinity chromatography. The antibodies may be purified so as to obtainspecific classes or subclasses of antibody such as IgM, IgG, IgA, IgG₁,IgG₂, IgG₃, IgG₄ and the like. Antibodies of the IgG class are preferredfor conferring passive immunity. The antibodies of the present inventionhave a number of diagnostic and therapeutic uses. The antibodies can beused as an in vitro diagnostic agents to test for the presence ofmicroorganisms in biological samples or in water or food samples, instandard immunoassay protocols. Such assays include, but are not limitedto, agglutination assays, radioimmunoassays, enzyme-linked immunosorbentassays, fluorescence assays, Western blots and the like. In one suchassay, for example, the sample is contacted with first antibodies of thepresent invention, and a labeled second antibody is used to detect thepresence of polysaccharides to which the first antibodies have bound.

Such assays may be, for example, of direct format (where the labeledfirst antibody is reactive with the polysaccharide), an indirect format(where a labeled second antibody is reactive with the first antibody), acompetitive format (such as the addition of a labeled polysaccharide),or a sandwich format (where both labeled and unlabeled antibody areutilized), as well as other formats describe in the art.

In providing the antibodies of the present invention to a recipientmammal, the dosage of administered antibodies will vary depending uponsuch factors as the mammal's age, weight, height, sex, general andspecific medical conditions, and the like.

In general, it is desirable to provide the recipient with a dosage ofantibodies which is in the range of from about 1 mg/kg to about 10 mg/kgbody weight of the mammal, although a lower or higher dose may beadministered. The antibodies of the present invention are intended to beprovided to the recipient subject in an amount sufficient to prevent, orlessen or attenuate the severity, extend or duration of the infection.

In order to facilitate the administration of the conjugates of theinvention to mammals, it is preferred that the conjugate be formulatedwith a pharmaceutically acceptable carrier. (Those skilled in the artwill appreciate the term “carrier,” when used in this context, has adifferent meaning that when it is used to refer to a biomoleculecomponent of the conjugate.) Examples of pharmaceutically acceptablecarriers include sterile water and saline, both of which may be bufferedwith phosphate, citrate, and the like. The conjugates of the inventionmay be provided in solution or suspension in a pharmaceuticallyacceptable carrier, or they may be provided in dry form andreconstituted with the pharmaceutically acceptable carrier prior toadministration.

The administration of the conjugates and compositions of the inventionmay be for prophylactic or therapeutic purposes. When providedprophylactically, the agents are provided in advance of any symptom. Theprophylactic administration of the agent serves to prevent or ameliorateany subsequent infection. When provided therapeutically, the agent isprovided at (or shortly after) the onset of a symptom of infection.

For all therapeutic, prophylactic and diagnostic uses, thepolysaccharide-carrier conjugates of this invention, as well asantibodies and other necessary reagents and appropriate devices anaccessories may be provided in kit form so as to be readily availableand easily used.

The examples below will be understood to be merely representative of theinvention, and are not intended to limit the scope of the appendedclaims in any way.

EXAMPLE 1

Dieneophile component 2: Treatment of human serum albumin (HSA) with a1.6 molar excess (based on 58 available amino groups) of3-sulfosuccinimidyl 4-maleimidobutyrate (1) in a pH 7.5 phosphate bufferafforded the intermediate 2, which contained an average of 38 maleimidomoieties per protein molecule, as indicated in the formula (determinedby MALDI-TOF mass spectroscopy).

Diene component 8: The phenylthio rhamnoside 3 was prepared as describedpreviously (V. Pozsgay, Carbohydr. Res. 1992, 235:295). Rhamnoside 3 wastreated with acetic anhydride and pyridine to afford 4, ¹H NMR (CDCl₃,δ) 8.11–7.26 (m, 15 H), 5.79 (dd, 1 H), 5.64–5.52 (m, 3 H) 5.53 (t, 1 H,J=10.0 Hz), 4.49 (dq, 1 H), 1.89 (s, 2 J=6.3 Hz), ¹³C (CDCl₃, δ) 170.1,165.7, 165.5, 133.5–127.9, 85.8, 72.1, 71.9, 69.5, 68.1, 20.6, 17.6.

From 4 the hemiacetal 5 was obtained by hydrolysis with mercurictribluoroacetate (L. Yan, D. Kahne, J. Am. Chem. Soc. 1996, 118:9239),¹H NMR (CDCl₃, δ) 8.1–7.4 (m, 10H), 5.71 (dd, 1 H, J=3.4 Hz, J=9.9 Hz),5.78 (dd, 1 H), 5.49 (t, 1 H, J=9.9 Hz), 5.38 (br d, 1 H), ¹³C (CDCl₃,δ) 170.3, 165.8, 92.2, 71.9, 71.0, 68.9, 66.7, 20.7, 17.7.

Hemiacetal 5 was converted to the trichloroacetimidate 6, andglycosylation of trasn,trans-hexa-2,4-dien-1-ol with 6 usingCF₃SO₃Si(CH₃)₃ as the activator afforded the glycoside 7. The acetylgroups were then removed by treatment with NaOMe to afford the dienerhamnoside 8. ¹H NMR (D₂O, δ) 6.33 (dd, 1 H, J=9.5 Hz, J=10.2 Hz), 6.16(ddd, 1 H, J=1.6 Hz, J=10.2 Hz, J=14.8 Hz), 5.85 (m, 1 H), 4.83 (d, 1 H,J=1.7 Hz), 4.21 (dd, 1 H, J=6.4 Hz, J=12.4 Hz), 4.07 (dd, 1 H, J=7.1 Hz,J=12.4 Hz), 3.91 (dd, 1 H, J=1.7 Hz, J=3.4 Hz), 3.69 (dq, 1 H), 3.73(dd, 1 H, J=3.4 Hz, J=9.6 Hz), H, J=9.6 Hz), 1.75 (d, 3 H, J=6.8 Hz),1.28 (d, 3 H, J=6.3 Hz), ¹³C (D₂O, δ) 136.7, 132.7, 131.1, 125.6, 99.8,72.8, 71.1, 71.0, 70.9, 69.4, 68.6, 18.2, 17.4.

Coupling reaction: An excess of 8 was treated in an aqueous solutionwith the maleimido-derivatized protein 2. The average incorporations ofthe hapten, as a function of time and temperature, are shown in Table 1.This data was obtained from the average molecular mass of the conjugatesdetermined by the MALDI-TOF method. As expected for a concertedcycloaddition reaction, the incorporation level depends on the reactiontime and temperature. At room temperature, approximately 63% of theavailable dienophile moieties in the protein participated in adductformation within 36 h, while at 40° C. almost complete utilization ofthese moieties occurred after four days (Table 1). The unreacted diene 8was recovered by diafiltration, and the conjugate 9 (free amino groupsnot shown) was then obtained as a white solid after freeze-drying.

TABLE 1 Time and temperature dependence of the cycloaddition between 2and 8 Composition of the conjugate (mol hapten/mol albumin) Time (h) 22°C. 40° C.  36 24 27 100 28 37

EXAMPLE 2

Dienophile component: The glycoside of 6-hydroxyhexanoic acid hydrazidewith the tetramer of(α-L-rhamnopyranosyl)-(1→2)-(α-D-galactopyranosyl)-(1→3)-(α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoseis prepared, according to the procedure disclosed in internationalpatent application WO 99/03871. Treatment with maleic anhydride providesan N-terminal maleimide derivative 10.

Diene component: Trans,trans-hexa-2,4-dien-1-ol and succinic anhydrideare reacted in the presence of N,N-dimethylaminopyridine to providetrans,trans-hexa-2,4-dien-1-ol monosuccinate. An aqueous solution of anexcess of the monosuccinate is activated with1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and coupled with humanserum albumin, to provide a poly(diene) derivative 11.

Coupling reaction: An aqueous solution of 11 and excess 10 is incubatedat 35° C. for 4 days, and the resulting conjugate 12 (n≦58) is purifiedby diafiltration and lyophilized. The conjugate 12 is expected to beuseful for inducing antibodies against Shigella dysenteriae.

EXAMPLE 3

Diene component: The Vi capsular polysaccharide of Salmonella typhi(Pasteur Merieux Serums et Vaccins, Lyon F R) is dissolved in water,excess 2,4-hexadienylamine is added, and the solution buffered to pH5.0. A slight excess of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimidein water is added. After 4 h at 37°, the mixture is adjusted to pH 7.5and dialyzed to remove excess reagents. The resulting solution of 13 isused immediately.

Dienophile component: Pseudomonas aeruginosa recombinant exoprotein A(Fattom el al., Infect. Immun. 1992, 60:584–589) is treated with excess3-sulfosuccinimidyl 4-maleimidobutyrate (1) as in Example 1, followed bydialysis and lyophilization to provide a maleimide derivatized protein14.

Coupling reaction: An excess of the diene 14 is added to the solution ofdienophile component 13. After 4 days at 37° C., the mixture isconcentrated, and conjugate 15 is purified by size exclusionchromatography on SEPHACRYL S-1000™ (Pharmacia, Piscataway N.J.). Theconjugate 15 is expected to be useful for inducing antibodies againstSalmonella typhi.

EXAMPLE 4

Diene component: To a solution of methyl octa-4,6-dienoate (16) (6 g) inmethanol (10 ml) was added hydrazine (3 ml) at room temperature. After24 h, the solution was diluted with water (50 ml). The crystallineprecipitate was isolated by filtration to afford octa-4,6-dienoic acidhydrazide (17) as colorless microcrystals, yield 5.7 g. Dextran (nominalMW 10 kDa, Pharmacia) was diafiltered through a YM10 membrane (MW cutoff10 kDa, Millipore) using 3 changes of water. The solution that passedthrough the membrane was diafiltered through a YM3 (MW cutoff 3000 Da)membrane, suing five changes of water. The material retained by themembrane was lyophilized. A stirred solution of the dextran 18 thusobtained (22.5 mg, 2.25 μmol, corresponding to 139 μmol of glucose) inH₂O (2.2 ml) at 5° C. (ice bath) was equipped with a temperature-sensingpH electrode, and 0.1 M NaOH was added with a 100 μL microsyringe untilthe pH reached 10.5. To this solution was added BrCN (50 μL of a 5 Msolution in acetonitrile). The pH of the solution was maintained between10.5 and 10.8 by addition of 0.1 M NaOH with a microsyringe. After 6min, the solution was adjusted to pH 8.5 by addition of pH 8.0 phosphatebuffer (ca. 1 ml). To the reaction mixture was immediately added asolution of octa-4,6-dienoic acid hydrazide (3.5 mg, 235 μmol) in 0.5 mldimethyl sulfoxide. The pH of the reaction mixture rose to 8.95, at atemperature of 9° C. The ice bath was removed and the stirred solutionwas allowed to reach 25° C. After 2.5 h, the pH was 8.18 and remainedunchanged over 5 min. The solution of 20 was diafiltered through a YM-3membrane using 5 changes of water (4 ml each).

Dienophile component: A stirred solution of human serum albumin (11.4mg, 0.17 μmol, 9.88 μmol of amino groups) in pH 7.5 buffer (1 ml) wastreated at 5° C. (ice-bath) with 3-sulfosuccinimidyl 4-maleimidobutyrate(1) (0.52 mg, 1.36 μmol). The solution was stirred for 15 min at 5° C.then for another 15 min at room temperature followed by diafiltrationthrough a YM-10 membrane using 5 changes of water (4 ml each).

Coupling reaction: The residual solutions containing the modifieddextran 20 and the modified human serum albumin were combined. The totalvolume of the combined solution was approx. 1 ml. After 22 h at roomtemperature, the reaction mixture was diafiltered through a YM-10membrane using 6 changes of H₂O (5 ml each), and the residue wasfreeze-dried. MALDI mass spectroscopy showed that most of the albumin isconsumed. The conjugate 21 that is formed has average molecular weightof 90 kDa.

EXAMPLE 5

Diene component: 6-Hydroxyhexanoic acid 23 (S. Sabesan, J. C. Paulson,J. Am. Chem. Soc., 1986, 108:2068) and glycosyl chloride 22 (V. Pozsgay,Glycoconj. J., 1993, 10:133), in methylene chloride at −40° C., weretreated with silver triflate and 2,6-di-t-butyl-4-methylpyridine for 10min. to afford glycoside 24. Deprotection with a catalytic amount ofNaOMe in MeOH at 23° C. for 24 hr, followed by treatment withethylenediamine in ethanol at 80° C. for 12 hr, provided 25 in 74%overall yield. ¹H NMR (500 MHz, CD₃OD): δ=4.64 (br s, 1 H), 3.78 (dd, 1H), 3.66 (m, 1 H), 3.55 (dq, 1 H), 3.39 (m, 1 H), 3.35 (t, 2 H), 2.76(t, 2 H), 2.21 (t, 2 H), 1.54–1.68 (m, 4 H), 1.35–1.46 (m, 2 H), 1.24 (d

Octadienoic acid 26 was prepared from the corresponding methyl ester (T.Hudlicky et al., J. Org. Chem., 1980, 45:5020). Acylation of 25 with 26(dicyclohexyl-carbodiimide, EtOAc, MeOH) afforded the glycoside diene 27in 92% yield, ¹H NMR (500 MHz, CD₃OD): δ=6.09 (m, 2 H), 5.70 (m, 1 H),5.57 (m,1 H), 4.77 (br s, 1 H), 3.92 (dd, 1 H), 3.63–3.75 (m, 4 H), 3.53(m, 1 H), 3.43 (t, 1 H), 3.32 (m, 4 H), 2.32 (m, 4 H), 2.22 (m, 2 H),1.72 (d, 3 H), 1.53–1.67 (m, 4 H), 1.30–1.42 (m, 2 H), 1.29 (d, 3 H),

Dienophile component: A stirred solution of human serum albumin in pH7.5 buffer (1 ml) was treated at 5° C. (ice-bath) with3-sulfosuccinimidyl 4-maleimidobutyrate (1) as described above, toprovide maleimido-derivatized protein containing an average of 29maleimido groups per molecule of protein.

Coupling reaction: The diene 27 was treated with themaleimido-derivatized protein in water at 23° C. Samples taken atvarious times were diafiltered through a 10 kDa cutoff membrane thenwere lyophilized and subjected to MALDI-TOF mass spectrometry. Theincrease in molecular weight of the resulting conjugate 28 (n≦29),relative to the mass of the core protein with the dienophiles attached,is shown as a function of time in FIG. 1.

A similar time-dependent experiment was conducted with the samemaleimido-derivatized protein, using previously described diene 8, andthe results are shown in FIG. 2. Kinetic parameters are shown in Table2.

TABLE 2 Kinetic Parameters for Conjugation Reactions Parameters for Δmw= Δmw_(max)(1 − e^(−kt)) Diene Δmw_(max) k (min⁻¹) t_(1/2) (min) 14 90000.007 99 8 4350 0.0045 154

EXAMPLE 6

Diene component: The previously describe ester 16 was converted tohydrazide 17 by treatment with hydrazine in MeOH at 23° C. for 24 hr.:¹H NMR (500 MHz, CD₃OD): δ=6.00 (m, 2 H), 5.57 (m, 1 H), 5.49 (m, 1 H),2.33 (m, 2 H), 2.21 ( (d, 3 H).

Acylation with methyl malonyl chloride in pyridine at −20° C. for 20 minafforded the ester 29 in 72% yield, ¹H NMR (500 MHz, CD₃OD): δ=6.03 (m,2 H), 5.58 (m, 2 H), 3.74 (s, 3 H), 3.41 (m, 2 H), 2.31–2.43 (m, 4 H),1.23 (d, 3 H).

Hydrolysis with LiOH in methanol (23° C., 1 hr), followed byacidification with 1 N HCl, gave the crystalline acid 30 in 74% yield,¹H NMR (500 MHz, CD₃OD): δ=5.97 (m, 2 H), 5.52 (m, 2 H), 3.26 (m, 2 H),3.24–3.37 (m, 4 H), 1.66 (d, 3 H).

Treatment of the dodecasaccharide hydraxzide 31 (V. Pozsgay, J. Org.Chem. 1998, 63:5983) with the linker 30 in DMF in the presence of HATUled to the diene-equipped construct 32, which was purified bygel-filtration through a Biogel P-4 column using water as the eluant. ¹HNMR (500 MHz, D₂O): δ=6.11 (m, 2 H), 5.72 (m, 1 H), 5.61 (m, 1 H), (brs, 3 H), 5.11 (br s, 2 H), 5.08 (br s, 1 H), 5.06 (br s, 2 H), 5.04 (d,2 H), 5.00 (br s, 1 H), 1.71 (d, 3 H); FAB-MS(dithiothreitol-dithioerythritol, positive ion) 2363.9 (M+Na), calcd.:2362.9.

Dienophile component: A stirred solution of human serum albumin in pH7.5 buffer (1 ml) was treated at 5° C. (ice-bath) with3-sulfosuccinimidyl 4-maleimidobutyrate (1) as described above, toprovide maleimido-derivatized protein containing an average of 22maleimido groups per molecule of protein.

Coupling reaction: Under conditions similar to those used for theconjugation of constructs 8 and 27, cycloaddition of 32 onto themaleimido-derivatized protein took place at a slower rate. After 2hours, the average incorporation was 1.5 dodecasaccharide chains per HSAmolecule, and after 8 hours, the incorporation reached an average of 3.

1. A conjugate of biomolecules of the formula

wherein R and R′ are independently H or methyl, or together constituteCH₂, CH₂CH₂, or O; X is CH or N; Y is N, CH═C, or NH—N; and B₁ comprisesa carbohydrate and B₂ comprises a peptide carrier, a protein carrier, ora polysaccharide carrier, or B₁ comprises a peptide carrier, a proteincarrier, or a polysaccharide carrier and B₂ comprises a carbohydrate;and wherein B₁ is attached at a ring position that is the same ringposition as R or R′ and B₁ and B₂ are each individually optionallyattached to the ring structure via a linker.
 2. The conjugate ofbiomolecules according to claim 1, wherein one of the biomolecules is apolysaccharide.
 3. The conjugate of biomolecules according to claim 2,wherein the polysaccharide is a viral or bacterial polysaccharide. 4.The conjugate of biomolecules according to claim 1, wherein one of thebiomolecules is a polysaccharide and the other biomolecule is apolypeptide.
 5. The conjugate of biomolecules according to claim 4,wherein the polysaccharide is a viral or bacterial polysaccharide.
 6. Animmobilized biomolecule of the formula

wherein R and R′ are independently H or methyl, or together constituteCH₂, CH₂CH₂, or O; X is CH or N; Y is N, CH═C, or NH—N; and B₁ comprisesa carbohydrate and B₂ comprises a peptide carrier, a protein carrier, ora polysaccharide carrier, or B₁ comprises a peptide carrier, a proteincarrier, or a polysaccharide carrier and B₂ comprises a carbohydrate;and wherein B₁ is attached at a ring position that is the same ringposition as R or R′ and B₁ and B₂ are each individually optionallyattached to the ring structure via a linker.
 7. A pharmaceuticalcomposition comprising a conjugate according to claim 1, furthercomprising a pharmaceutically acceptable carrier.
 8. The conjugate ofclaim 1 wherein the B₁ or B₂ carbohydrate comprises an oligosaccharideor a polysaccharide.
 9. The conjugate of claim 1 wherein the B₁ or B₂carbohydrate comprises a hapten or an antigen.
 10. The conjugate ofclaim 1 wherein the B₁ or B₂ carbohydrate comprises a polysaccharidehapten or antigen.
 11. The immobilized biomolecule of claim 6 whereinthe B₁ or B₂ carbohydrate comprises an oligosaccharide or apolysaccharide.
 12. The immobilized biomolecule of claim 6 wherein theB₁ or B₂ carbohydrate comprises a hapten or an antigen.
 13. Theimmobilized biomolecule of claim 6 wherein the B₁ or B₂ carbohydratecomprises a polysaccharide hapten or antigen.